In recent years, structural and functional analyses of proteins in living tissues have been rapidly promoted as the post-genome research. For such structural and functional analyses of proteins (proteome analysis), the methods that have involved using a mass spectrometer for the expression analysis or primary structure analysis of a protein have been widely used in recent years. A conventional example of this kind of method is called a peptide mass fingerprinting (PMF). This method includes extraction of a protein from a sample, followed by the purification and separation of that protein by two-dimensional electrophoresis or other techniques. The separated protein is then digested with an appropriate enzyme to form a mixture of peptide fragments, and the mass of each fragmentary peptide is precisely measured by a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS) or other apparatuses. Subsequently, a database search using a search engine is performed to locate a protein containing a peptide whose mass matches the measured mass data. Eventually, a list of probable candidates of the protein is shown as an identification result.
A mass spectrometer capable of MS/MS analysis (or tandem analysis), which captures and dissociates a specific peak by a quadrupole ion trap, collision-induced dissociation (CID) and other techniques, can also be used to determine the amino acid sequence of a protein as follows: The protein is initially digested with an appropriate enzyme to form a mixture of peptide fragments. This peptide mixture is then subjected to mass analysis. The constituent elements of each peptide have stable isotopes having different masses. Therefore, even if one peptide has the same amino acid sequence as that of another, these peptides will have peaks at different mass-to-charge ratios due to the difference in their isotopic composition. The resultant peaks include the peak of an ion that has no isotopic element (mono-isotopic ions) and those of the other ions that contain isotopic elements (isotopic ions). These peaks form a peak group in which a plurality of peaks are located at intervals of 1 Da (in the case of a monovalent ion). (This group is hereafter called an isotopic peak group.)
Subsequently, one isotopic peak group originating from the same peptide is entirely (or partially in some cases) selected as a precursor ion from the mass spectrum data of the peptide mixture. This precursor ion is then dissociated into smaller ions (product ions), which in turn are subjected to mass analysis (MS/MS analysis). Based on the pattern of the mass spectrum of the product ions obtained in this manner and/or the pattern of the mass spectrum of the precursor ion, a database search is conducted to determine the amino acid sequence of the peptide in question and identify the protein (refer to Patent Document 1 and other references).
The previously described protein identification technique basically premises the extraction of proteins from a cell or other specimen, followed by the purification and separation of the proteins to prepare a sample to be analyzed. However, in biochemical, medical and other technical fields, there is a strong demand for the acquisition of information relating to a two-dimensional protein distribution within an in-vivo cell without breaking the cell. To fulfill this requirement, a mass microscope having the functions of both a microscope and a mass spectrometer has been under development. (A mass microscope may also be called an imaging mass spectrometer). With an imaging mass spectrometer, it is possible to obtain distribution information (e.g. a mapping image) of a substance within a two-dimensional area on a sample placed on a preparation or other locations. To date, various configurations have been proposed for the acquisition of mass spectrum data for each micro area within a two-dimensional area on a sample in an imaging mass spectrometer.
For example, the mass spectrometer disclosed in Patent Document 2 or Non-Patent Document 1 scans the surface of a sample by sequentially moving the irradiation point of a laser beam or particle beam for ionization; every time the irradiation point is moved, the ions generated from the irradiation point are individually detected with respect to their mass. According to the method proposed in Non-Patent Document 2, ions are almost simultaneously generated over a two-dimensional area of a sample so that they will reflect the two-dimensional distribution of the substances on the sample; those ions are then mass-separated by a time-of-flight mass separator and detected by a two-dimensional detector.
In any of these techniques, creation of a mapping image of a substance within a two-dimensional area on a sample requires analyzing and processing mass spectrum data obtained for each micro area within the two-dimensional area and specifying a substance (e.g. a protein) present in each micro area. In the case of a mass spectrometer capable of MS/MS analysis (or tandem analysis), mass spectrum data obtained by a mass analysis without dissociation of ions is initially analyzed to specify an ion to be selected as a precursor ion. After an appropriate precursor ion is selected for each micro area, an MS/MS analysis is carried out to obtain mass spectrum data for each micro area, and these data are analyzed to identify a substance present in each micro area.
However, it is difficult to identify a protein within a micro area on a sample on the basis of mass spectrum data obtained for that area. A major reason is because there are often two or more kinds of proteins within one micro area if the sample is a portion of a living tissue. In the case of the aforementioned protein identification technique, it is least possible for peaks originating from different proteins to overlap each other on a mass spectrum since each protein contained in an analyte is purified and separated beforehand by a preprocess. On the other hand, performing a mass analysis of a sample with two or more kinds of proteins mixed together allows peaks originating from different proteins to be mixed together on the mass spectrum. In this state, it is impossible to correctly identify each protein by referencing a database prepared for the deduction of amino acid sequences. Another problem with this type of mass spectrum exists in that locating an isotopic peak group in the previously described manner is difficult since two or more isotopic peak groups originating from different proteins are often overlapped with each other, making it difficult to correctly identify the peaks belonging to a specific isotopic peak group by simply checking their peak intensity ratio. This results in a decrease in the identification accuracy since a set of peaks originating from the same substance cannot be correctly selected as a precursor ion for the MS/MS analysis.    Patent Document 1: Japanese Unexamined Patent Application Publication No. 2006-284509    Patent Document 2: U.S. Pat. No. 5,808,300    Non-Patent Document 1: Kiyoshi Ogawa et al., “Kenbi Shisuryou Bunseki Souchi No Kaihatsu (Research Development of Mass Microscope)”, Shimadzu Hyouron (Shimadzu Review), Shimadzu Hyouron Henshuu-bu, Mar. 31, 2006, Vol. 62, No. 3/4, pp. 125-135    Non-Patent Document 2: Yasuhide Naito, “Seitai Shiryou Wo Taishou Ni shia Shitsuryou Kenbikyou (Mass Microprobe Aimed at Biological Samples), Journal of the Mass Spectrometry Society of Japan Vo. 53 (2005), No. 3